Rapamycin 40-O-Cyclic Hydrocarbon Esters, Compositions and Methods

ABSTRACT

A new class of rapamycin 40-O-cyclic hydrocarbon esters is disclosed. The 40-O position of the rapamycin ester has the form 40-O—R, where R is C(O)—(CH 2 ) n —X, n is 0, 1 or 2, and X is a cyclic hydrocarbon having 3-8 carbons, optionally containing one or more unsaturated bonds, and one or more linear (CH 2 ) n ) and/or cyclic (X) carbon atoms may have an OH or halide group. Also disclosed are therapeutic compositions and methods that employ the novel analogs.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.15/829,012, filed Dec. 12, 2017, which is a divisional application ofU.S. application Ser. No. 15/212,987, filed Jul. 18, 2016, now U.S. Pat.No. 9,867,911, which is a continuation application of U.S. applicationSer. No. 14/406,198, filed Dec. 5, 2014, now U.S. Pat. No. 9,408,884,which is a § 371 application of International Application Serial No.PCT/EP2013/061343 filed Jun. 3, 2013, which claims the benefit ofpriority from U.S. Provisional Application Ser. No. 61/657,049, filedJun. 8, 2012.

The present invention relates to a rapamycin 40-O-cyclic hydrocarbonester, compositions containing the ester, and methods of use of thecompound and composition.

Rapamycin is a macrocyclic triene compound that was initially extractedfrom a streptomycete (Streptomyces hygroscopicus) isolated from a soilsample from Easter Island (Vezina et al., J. Antibiot. 28:721 (1975);U.S. Pat. Nos. 3,929,992; 3,993,749).

A variety of rapamycin derivatives designed to improve its solubility,stability, and/or pharmacological profile have been reported. See, forexample, Adamczyk et al. Lipase Mediated Hydrolysis of Rapamycin42-Hemisuccinate Benzyl and Methyl Esters, Tetrahedron Letters, Vol. 35,No. 7, pp. 1019-1022, 1994, and U.S. Pat. Nos. 5,258,389, 5,665,772, and6,440,990.

One of the major therapeutic uses of rapamycin and its derivatives hasbeen for the treatment of restenosis. Restenosis after percutaneoustransluminal coronary angioplasty (PTCA) remains a major limitation(Hamon, M. et al., Drug Therapy, 4:291 301 (1998)). In a typical PTCAprocedure, the region of vascular blockage is expanded by balloonangioplasty and a stent is expanded against the vessel walls to maintainthe vessel in an expanded diameter state. With a bare metal stentrestenosis of the vessel may occur within 3-6 months or more in morethan 30% of the cases, requiring additional intervention to restore thevessel to an expanded diameter. Restenosis after PTCA is thought to be atwo-component process of both intimal hyperplasia and vascularremodeling, the former coming initially, the latter occurring later inthe process (Hoffman, R. et al., Circulation, 94:1247 1254 (1996);Oesterle, S. et al., Am. Heart J., 136:578 599 (1998)).

To reduce the rate of restenosis in a PTCA procedure, the stent may becoated with rapamycin or a rapamycin derivative in a form that allowsslow release of the drug from the stent against the endothelial cells ofthe vessel, typically over a two-week to several-month interval. Thestent coating may consist of a polymer, e.g., bioerodable polymer, withencapsulated drug, or the drug itself may form a cohesive coating. Ineither case, the coating may be susceptible to cracking as the stent isexpanded at the implantation site, and any loose pieces of coating thatbreak off can be a significant clotting hazard in the bloodstream.Another problem that has been observed heretofore in rapamycin stents isrelatively poor drug stability, as evidence by mass balance measurementon the amounts of active drug released from the stent coatings,typically showing less than 40% of active drug recovered after elutionfrom the stent.

In one aspect, the invention includes a rapamycin 40-O-cyclichydrocarbon ester having the structure:

where R is C(O)—(CH₂)_(n)—X, n is 0, 1 or 2, X is a cyclic hydrocarbonhaving 3-8 carbons, optionally containing one or more unsaturated bonds,one or more linear-chain or cyclic carbon atoms may contain an OH orhalide substitution, and the rapamycin parent structure may containsubstitutions at other positions.

Exemplary R groups include:

In another aspect, the invention includes a method of treating any ofthe following conditions in a mammal: (i) restenosis; (ii) woundhealing; (iii) vascular injury; (iv) vascular inflammation; (v)transplantation rejection; (vi) proliferative ophthalmic diseases,including wet age-related macular degeneration (AMD) and diabeticmacular edema (DME); (vii) uveitis; (viii) cancer; (ix) various skinconditions such asatopic dermatitis, eczema, tuberous sclerosis,neurofibromatosis, lichen planus and the like; (x) ear-nose-throattreatments such as sinusitis treatment with sinus stents or balloons byadministering to the mammal, a therapeutic amount of the rapamycin40-O-cyclic hydrocarbon ester. For inhibiting restenosis at a vascularinjury site, the rapamycin ester may be administered from a drug-elutingstent placed at the vascular injury site.

With respect to treating uveitis in a mammal, it is known in the artthat uveitis is a form of eye inflammation that affects the uvea, whichis the middle fibrous protective layer of tissue in the eye wall.Possible causes of uveitis are infection, acute injury, or autoimmunedisorder. Due to the specific location of the uvea, treatment regimenshave been notorious difficult and, if uveitis is left untreated, thedisease can progress to partial vision loss and even total blindness. Inone aspect, the present invention provides for a method of treatinguveitis in a mammal, comprising applying an effective amount of therapamycin 40-O-cyclic hydrocarbon ester of the present inventiondirectly to an eye of the mammal.

With respect to treating wet age-related macular degeneration in amammal, it is well-known that AMD is a progressive eye condition that isconsidered the most common cause of blindness in developed countries.AMD causes damage to the retina, with the most prevalent form of thedisease occurring when blood vessel growth is hyperstimulated by theprotein vascular endothelial growth factor (VEGF). While there are knownantibody treatments (anti-VEGF) available, such formulations requirefrequent dosing and may cause some reversion to a more recalcitrant formof AMD. In one aspect, the present invention provides a method oftreating AMD in a mammal, the method comprising applying an effectiveamount of the rapamycin 40-O-cyclic hydrocarbon ester of the presentinvention directly to an eye of the mammal.

Also disclosed is a drug-eluting stent having an expandable stent bodyformed of one or more filaments and carried on the one or morefilaments, a coating containing the above rapamycin 40-O-cyclichydrocarbon ester. The coating may be formed of the rapamycin compoundalone, or formed of a polymer, e.g., bioerodable polymer such aspolylactic acid, containing the rapamycin ester in entrapped form. Othersuitable polymers include poly(caprolactone), poly(trimethylenecarbonate), polyester amide, poly(glycolide), polyhydroxyalkanoatesincluding poly(hydroxyvalerate), poly(hydroxybutyrate),poly(hydroxybutyrate-co-valerate), polyorthoester, polyanhydride,poly(glycolic acid). The stent is used in a method for inhibitingrestenosis at the site of vascular injury, by placing the stent at thesite of vascular injury.

One specific aspect of the invention is directed to a method of treatingproliferative ophthalmic diseases, including wet age-related maculardegeneration (AMD) and diabetic macular edema (DME), and uveitis byadministering to the mammal, a therapeutic amount of the rapamycin40-O-cyclic hydrocarbon ester by way of an intravitreal injection of aformulation comprising or consisting of ethanol and the rapamycin40-O-cyclic hydrocarbon ester.

Further disclosed are pharmaceutical compositions, includingmicroparticle compositions, such as liposomes and bioerodable polymerparticle compositions, containing the rapamycin ester in an encapsulatedor captured form for release over time from a site of administration orimplantation of the composition. Microparticles containing the rapamycinanalog may be inherently porous with pore sizes ranging from about 5nanometers to about 10 microns in diameter.

In still another embodiment, the invention includes an improvement in anintravascular stent of the type having an expandable stent body formedof one or more filaments, such as a metal-filament stent, and carried onthe one or more filaments, a polymer coating containing 40%-80% polymer,such as a bioerodable polymer, and 20% to 60% rapamycin compound havinga 40-position —OH or —O(CH)_(n)OH substituent, where n=1 to 5. Theimprovement, which significantly reduces the tendency of the coating toflake or crack when the stent body is expanded, includes substituting inthe rapamycin compound, the rapamycin 40-O-cyclic hydrocarbon ester ofthe invention.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying figures.

FIG. 1 shows the effects of stent expansion on a polymer coatingcontaining 50 weight percent rapamycin on the stent filaments;

FIG. 2 shows the effects of stent expansion on a polymer coatingcontaining 50 weight percent rapamycin 40-O-cyclic hydrocarbon ester inaccordance with one aspect of the invention.

FIG. 3 shows a reverse phase HPLC comparison of drug breakdown materialsafter 72 hours incubation in rabbit vitreous humour at 37° C. (A) CRC;(B) sirolimus.

FIG. 4 shows results from a cell inhibition assay measuring the IC₅₀ ofthe rapamycin 40-O-cyclic hydrocarbon ester compounds of the presentinvention when applied to human breast cancer cell line MCF7.

FIG. 5 shows results from a cell inhibition assay measuring the IC₅₀ ofthe rapamycin 40-O-cyclic hydrocarbon ester compounds of the presentinvention when applied to human breast cancer cell line BT549.

FIG. 6 shows results from a cell inhibition assay measuring the IC₅₀ ofthe rapamycin 40-O-cyclic hydrocarbon ester compounds of the presentinvention when applied to human melanoma cell line A375.

FIG. 7 shows results from a cell inhibition assay measuring the IC₅₀ ofthe rapamycin 40-O-cyclic hydrocarbon ester compounds of the presentinvention when applied to human colorectal adenocarcinoma cell lineHT29.

FIG. 8 shows results from a cell inhibition assay measuring the IC₅₀ ofthe rapamycin 40-O-cyclic hydrocarbon ester compounds of the presentinvention when applied to human colorectal adenocarcinoma cell lineWiDr.

FIG. 9 shows results from a cell inhibition assay measuring the IC₅₀ ofthe rapamycin 40-O-cyclic hydrocarbon ester compounds of the presentinvention when applied to human pancreas ductal adenocarcinoma cell linePANC-1.

FIG. 10 shows results from a cell inhibition assay measuring the IC₅₀ ofthe rapamycin 40-O-cyclic hydrocarbon ester compounds of the presentinvention when applied to human pancreas ductal adenocarcinoma cell lineMiaPaCa2.

I. DEFINITIONS

“Rapamycin” refers to the macrocyclic triene compound having thestructure shown below, where the 40 position is the ring carbon positionshown below to which an OR group is attached, where R is H forrapamycin. Although not shown, the term “rapamycin” is also intended toencompass rapamycin analogs having substitutions at positions other thanthe 40-position, such as the non-position-40 substitutions andmodifications shown in U.S. Pat. Nos. 4,316,885, 4,650,803, 5,102,876,5,138,051, 5,118,678, 5,118,677, 5,100,883, 5,151,413, 5,120,842,5,120,725, 5,120,727, and 5,310,903, 5,665,772, and 6,440,990.

A “rapamycin 40-O-cyclic hydrocarbon ester” refers to a rapamycin analoghaving a 40-position —OR, where R is C(O)—(CH₂)_(n)—X, n is 0, 1 or 2, Xis a cyclic hydrocarbon having 3-8 carbons, optionally one or moreunsaturated bonds, and one or more linear (CH₂)_(n)) and/or cyclic (X)carbon atoms may have an OH or halide group. Optionally, the rapamycinparent structure may include substitutions at positions other than the40-0 position, such as described above.

II. SYNTHESIS OF RAPAMYCIN 40-O-CYCLIC HYDROCARBON ESTERS

Table I below shows the R group for a number of exemplary analogs formedin accordance with the present invention.

TABLE I

1

2

3

4

5

6

7

As detailed below, compounds 1-4 and 7 can be prepared by reactingrapamycin with the carbonyl chloride of the ester R group to be added,where the carbonyl chloride is readily prepared by known methods orobtained commercially. Compounds 5 and 6, in which R has one or more OHsubstituents, can be more easily prepared by reacting rapamycin with thefree acid of the desired R group in the presence of carbodiimide, thusavoiding the somewhat complicated chemistry of forming a carbonylchloride in the presence of a free OH group. It will be appreciated fromthese synthetic schemes how other R groups, e.g., where R is a seven- oreight-member cyclic ring, with or without OH or halide substituents, canbe prepared.

IIA. Rapamycin 40-O-Cyclic Hydrocarbon Ester with CyclopropanecarbonylChloride (Compound 1)

Compound 1 was prepared by dissolving 200 mg rapamycin in 1.0 mLmethylene chloride contained in a 3.8-mL borosilicate glass vial withstirring bar and capped. With stirring, 30 μL pyridine was addedfollowed with 100 μL cyclopropanecarbonyl chloride, as received. Thereaction vessel was placed at ambient temperature and reaction continuedwith stirring for 1 hour. The reaction mixture was transferred into a125-mL separatory funnel with a Pasteur pipette. 50 mL of ethyl acetatewas added to the reaction solution and the resulting solution washedwith 6 mL 1N HCl combined with 30 mL DI water. The organic phase wascollected into a beaker and the remaining aqueous layer wasback-extracted with 20 mL ethyl acetate. The acid washed organic phaseswere combined and further washed with 50 mL NaCl brine until a pH of 7was reached by litmus paper. The solution was then dried using anhydroussodium sulfate and concentrated under vacuum to result in a light yellowoil.

The product was purified using a CombiFlash system via normal phasechromatography. RediSep Rf 24 g Gold silica column was used with ahexanes/ethyl acetate solvent system. Purification started at 80/20hexanes/ethyl acetate for the first 20 minutes and ramped up to a 50/50hexanes/ethyl acetate mobile phase over a period of 27 minutes. Asdetermined by HPLC analyses, fractions containing the ester product werecombined and concentrated under vacuum to yield approximately 50% of thetheoretical. The purified product was reconstituted in 280 μL methanoland precipitated into 2.6 mL cold DI water. The white solid was filteredand dried in a vacuum oven held at 45° C. and under a pressure of −28.0in Hg for 24 hours.

The product was analyzed by High Resolution LCMS. Table II compares theexact mass calculated and exact mass found for the title compound(C₅₅H₈₃NO₁₄) with Na⁺ adduct. Also shown is dried product recovery.

TABLE II C₅₅H₈₃NO₁₄Na Exact Mass Calculated 1004.5711 Exact Mass Found1004.5696 Recovery Sirolimus Starting Material 200 mg Amount of ProductRecovered  80 mg

IIB. Rapamycin 40-O-Cyclic Hydrocarbon Ester with CyclobutanecarbonylChloride (Compound 2)

Compound 2 was prepared by the same scheme for Compound 1, butsubstituting 100 μL cyclobutanecarbonyl chloride, as received, for 100μL cyclopropropanechloride. The compound was purified as described forCompound 1 and analyzed by High Resolution LCMS.

Table III compares the exact mass calculated and exact mass found forthe title compound (C₅₆H₈₅NO₁₄) with Na⁺ adduct. Also shown is driedproduct recovery.

TABLE III C₅₆H₈₅NO₁₄Na Exact Mass Calculated 1018.5868 Exact Mass Found1018.5791 Recovery Sirolimus Starting Material 200 mg Amount of ProductRecovered  73 mg

IIC. Rapamycin 40-O-Cyclic Hydrocarbon Ester with CyclopentanecarbonylChloride (Compound 3)

Compound 3 was prepared by the same scheme for Compound 1, butsubstituting 100 μL cyclopentane chloride, as received, for 100 μLcyclopropanecarbonyl chloride. The compound was purified as describedfor Compound 1 and analyzed by High Resolution LCMS.

Table IV compares the exact mass calculated and exact mass found for thetitle compound (C₅₇H₈₇NO₁₄) with Na⁺ adduct. Also shown is dried productrecovery.

TABLE IV C₅₇H₈₇NO₁₄Na Exact Mass Calculated 1032.6024 Exact Mass Found1032.5951 Recovery Sirolimus Starting Material 200 mg Amount of ProductRecovered  84 mg

IID. Rapamycin 40-O-Cyclic Hydrocarbon Ester with CyclohexanecarbonylChloride (Compound 4)

Compound 4 was prepared by the same scheme for Compound 1, butsubstituting 100 μL cyclohexanecarbonyl chloride, as received, for 100μL cyclopropanecarbonyl chloride. The compound was purified as describedfor Compound 1 and analyzed by High Resolution LCMS.

Table V compares the exact mass calculated and exact mass found for thetitle compound (C₅₈H₈₉NO₁₄) with Na⁺ adduct. Also shown is dried productrecovery.

TABLE V C₅₈H₈₉NO₁₄Na Exact Mass Calculated 1046.6181 Exact Mass Found1046.6123 Recovery Sirolimus Starting Material 200 mg Amount of ProductRecovered 122 mg

IIE. Rapamycin 40-O-Cyclic Hydrocarbon Ester with 4-HydroxycyclohexaneCarboxylic Acid (Compound 5)

Compound 5 was prepared by dissolving 9 μL mg rapamycin in 2.5 mLmethylene chloride in a 7.5-mL borosilicate glass vial with a stir barand capped. The solution was stirred at ambient temperature for twominutes, then 78.0 mg 4-N,N-dimethylaminopyridine was added and allowedto dissolve. 118 mg racemic 4-hydroxycylohexane carboxyl acid and 204 mgN,N-dicyclohexylcarbodiimide were added in the reaction mixture. Thereaction continued stirring at ambient temperature for 2 hours.Synthesis of product was verified by taking 2 μL of the reaction mixtureand diluting in 0.5 mL acetonitrile and analyzed by High ResolutionLCMS. Table VI compares the exact mass calculated and exact mass foundfor the title compound (C₅₈H₈₉NO₁₅) with Na⁺ adduct.

TABLE VI C₅₈H₈₉NO₁₅Na Exact Mass Calculated 1062.6130 Exact Mass Found1062.6130

IIF. Rapamycin 40-O-Cyclic Hydrocarbon Ester with Shikimic Acid(Compound 6)

Compound 6 was prepared by dissolving 14.0 mg rapamycin in 100 μLmethylene chloride contained in a 1.5-mL HPLC glass vial with stirringbar and capped. The solution was stirred at ambient temperature for twominutes, then 1.0 mg 4-N,N-dimethylaminopyridine was added. In a second1.5-mL HPLC glass vial, 2.0 mg shikimic acid and 100 μLdimethylformamide were combined and mixed. Using a syringe, the shikimicacid/dimethylformamide solution mixture was added dropwise into therapamycin solution. 3.0 mg N,N-dicyclohexylcarbodiimide was added andthe reaction was continued with stirring for 19 hours at ambienttemperature. Synthesis of product was verified by taking 2 μL of thereaction mixture and diluting in 0.5 mL acetonitrile and analyzed byHigh Resolution LCMS. Table VII compares the exact mass calculated andexact mass found for the title compound (C₅₈H₈₇NO₁₇) with Na⁺ adduct.

TABLE VII C₅₈H₈₇NO₁₇Na Exact Mass Calculated 1092.5872 Exact Mass Found1092.5850

IIG. Rapamycin 40-O-Cyclic Hydrocarbon Ester with CyclohexaneacetylChloride (Compound 7)

Compound 7 was prepared by dissolving 20 mg rapamycin in 100 μLmethylene chloride contained in a 1.5-mL HPLC glass vial with stirringbar and capped. With stirring, 3 μL pyridine was added and followed with10 μL cyclohexaneacetyl chloride, as received. The reaction vessel wasplaced at ambient temperature and reaction continued with stirring for 1hour.

The product was analyzed by High Resolution LCMS. Table VIII comparesthe exact mass calculated and exact mass found for the title compound(C₅₉H₉₁NO₁₄) with Na⁺ adduct.

TABLE VIII C₅₉H₉₁NO₁₄Na Exact Mass Calculated 1060.6337 Exact Mass Found1060.6337

III. COMPOUND PROPERTIES IIIA. Increased Ductility of a Coating Formedfrom the Analog

The 40-O-cyclic hydrocarbon rapamycin esters of the invention aredesigned for use in a variety of therapeutic uses, as detailed inSection IV below. One of the important uses is for slow-release of thedrug from a stent coating in treating/preventing restenosis followingpercutaneous transluminal coronary angioplasty (PTCA). In thisprocedure, the coated stent is delivered to the site of vascular injuryin a contracted, low-diameter condition in a balloon catheter, and thenexpanded to the dimensions of the treated vessel at the site. In thisapplication, it is important that the drug coating is sufficientlyductile that it does not crack or flake off the outer stent surfaceduring stent expansion, since loose pieces of coating, if released intothe bloodstream, may present a serious clotting hazard.

In accordance with one of the unexpected advantages of the presentinvention, it has been discovered that a coating formed with thecompounds of the present invention is substantially more ductile, i.e.,less prone to cracking, than a coating formed with rapamycin. Thisproperty can be seen from the photomicrographs of FIGS. 1 and 2, whichshow the condition of a stent coating after stent expansion in a stentformed of a 1:1 weight ratio of a 40-O-cyclic hydrocarbon rapamycinester and polylactic acid (FIG. 1) and a coating formed of a 1:1 weightratio of Compound 1 (Table I) and polylactic acid (FIG. 2). As seen, thedrug compound of the present invention provides for a coating that ismore ductile, and more resistant to delamination and particle sheddingand in general, appears to function as a plasticizer for the polylactatecoating. An important advantage of the drug compound in this setting isthat a coating with a high drug:polymer ratio can be prepared withoutthe need for addition of a separate plasticizer, which may cause toxicresponses, affect drug elution rates, and extend development times andincrease production costs.

In one aspect, the invention includes an improvement in an intravascularstent of the type having an expandable stent body formed of one or morefilaments, such as a metal-filament stent, and carried thereon, adrug-eluting polymer coating containing 40-80 weight percent polymer and20 to 60 weight percent of a rapamycin compound having a 40-O—H or40-O-linear alcohol group. The improvement, which significantly reducesthe tendency of the coating to flake or crack when the stent body isexpanded, includes substituting for the rapamycin compound, therapamycin 40-O-cyclic hydrocarbon ester of the present invention.

IIIB. Drug Elution and Stability

This section examines the stability of the compounds of the inventionwhen released from a reservoir over a several-day to several-weekperiod. In one study, a series of stents were prepared with a suitablecoating of drug/polymer mixture. The stents were fabricated from lasercut and electropolished 316 L Stainless Steel and measured 16millimeters in length. The coating mixture contained an equal weight ofpolylactic acid and each of the compounds to be evaluated. The mixturecomponents were weighed, combined in a glass vial with acetone at roomtemperature and then vortexed until all of the materials dissolved. Eachindividual stent was weighed using a Mettler model XP105 balance. AHamilton brand micro syringe with a capacity of 25 microliters and a 26gauge dispensing needle was used to aspirate a column of the coatingfluid from the glass vial. Each stent was held on a coating mandrellocated under a stereo zoom microscope at 60× magnification.

The coating was applied to the outer surface of each of the stents bymeans of mounting the syringe into a KD Scientific model 100 syringepump and then gradually metering the solution onto the stent struts in auniform manner. After coating each of the stents was vacuum dried atroom temperature under vacuum (25 inches Hg) for a period of 72 hours.The weight of the drug coating was confirmed by re-measurement of eachstent and subtraction of the weight of the bare stent from the coatedstent. The target weight of the coating was 500 micrograms which yieldeda drug dose of 250 micrograms for each of the coated stents. Each stentwas also visually inspected to confirm the integrity of the coating.

After visual inspection the stents were mounted onto 3.0 mm×16 mmangioplasty balloons using a hand crimper. The coated stents weresubsequently deployed to a nominal diameter of 3.0 mm using a Braun Co.indeflator device filled with deionized water. The appropriate pressurefor inflation was determined using the inflation table applicable to theangioplasty catheter. Upon balloon deflation, the stent was carefullyremoved and placed into a glass vial which was capped and saved for theelution analysis.

For elution comparison each stent was transferred into its respective7-mL borosilicate vial and 4.0 mL 25% ethanol in water (w/w) elutionmedium was added. Each vial was capped and incubated statically in a 37°C. water bath. At specific time points, the elution medium was removedfrom each vial for analysis and replenished with 4.0 mL fresh elutionmedium. The amount of drug eluted during each time interval wasdetermined by HPLC. After a cumulative incubation of 168 hours, theremaining drug in each stent was determined by exhaustive extraction byadding 4.0 mL acetonitrile to each stent for 20 minutes at ambienttemperature followed by vortexing for 10 seconds. These conditionsdissolve all drug and polymer remaining on the stent. Measurement ofdrug content from each exhaustive extraction was determined by directanalysis of the acetonitrile using HPLC as above. The amount of drugrecovered at each time point and the exhaustive extraction was summedfor each stent to determine the total amount of drug recovered, and theresults for rapamycin and a rapamycin 40-O-cyclic hydrocarbon ester areshown in Table IX below.

TABLE IX Total Drug Drug (μg) Stent # Amount (μg) Recovered % RecoveryRapamycin 1 238 84.6 35.6 2 238 103.4 43.4 3 236 125.2 53.1 4 236 71.632.8 5 241 109.1 45.3 Average 42.0 Standard Deviation 8.1 Rapamycin 40-6 228 192.5 84.6 Cyclic 7 233 202.2 87.0 Hydrocarbon 8 233 198.7 85.3Ester 9 236 202.4 85.8 10 241 197.8 82.2 Average 85.0 Standard Deviation1.8

Average % recovery for rapamycin and the rapamycin 40-cyclic hydrocarbonester were 42.0±8.1 and 85.0±1.8, respectively. This indicates that,unexpectedly, drug compounds of the present invention are more stablethan rapamycin during elution conditions. This property is important asit allows for a lower drug dose to be utilized for patient treatment andalso is beneficial for the drug product regulatory approval and qualitycontrol purposes because a high in vitro elution recovery is taken asevidence that the drug substance is stable, the test procedure is anaccurate monitor of manufacturing quality and good drug productperformance can be maintained.

IIIC. Inhibition of Restenosis Trial A

Stainless steel coronary artery stents coated with the rapamycin40-O-cyclic hydrocarbon ester of the invention were compared for theirability to control induced restenosis using a 28 day porcine arterialover stretch model. Four different groups of stents were evaluated withthe drug compound and in different coating configurations. Group 1consisted of five stainless steel stents which were first coated with abase layer consisting of a copolymer of polycaprolactone and poly Llactic acid. A topcoat of poly d,l lactic acid and 225 micrograms ofrapamycin 40-O-cyclic hydrocarbon ester was then applied. Group 2consisted of five stainless steel stents with the same base coat and atopcoat of poly d,l lactic acid and 100 micrograms rapamycin 40-O-cyclichydrocarbon ester. Group 3 consisted of five stents with a drug topcoatconsisting only of 100 micrograms of rapamycin 40-O-cyclic hydrocarbonester applied directly to a base coat of the polycaprolactone-poly Llactic acid copolymer. Finally, Group 4 consisted of five stents with abase coat of parylene polymer and a topcoat of poly d,l lactic acid and225 micrograms of rapamycin 40-O-cyclic hydrocarbon ester. All of thestents were 16 millimeters in length and were mounted on standardballoon delivery systems and were then sterilized with ethylene oxidegas. The balloon to artery ratio of the deployed stents wasapproximately 1.20:1.00. The matrix of coating layers and drug dosageswas chosen with the intent to optimize the adhesion of the active drugcompound to the stent surface and to determine the best means totransfer the drug to the surrounding vessel wall upon stent deployment.

The primary objective of this study was to evaluate the drug compoundcoating on stents to treat vessels in which injury was intentionallyinduced. Coronary artery measurements were made from angiographic imagesbefore stent deployment, immediately after deployment, and twenty-eightdays after deployment to determine the effectiveness of the coated stentin reducing restenosis. Determination of angiographic late loss was madeby subtraction of the minimum vessel lumen diameter of the stentedvessel at 28 days post-implant from the minimal vessel lumen diameter atthe time of implant. Determination of percent diameter stenosis was madeat 28 days post-implant by the calculation of 100(1-MLD/refdia.) whereMLD represents the minimum vessel lumen diameter at 28 days post-implantand refdia. is the average diameter of the vessel sections adjacent tothe stented vessel. In prior porcine studies, vessels with implanteduncoated stainless steel stents indicated mean angiographic late lossvalues of one millimeter or more and mean angiographic percent diameterstenosis values of approximately 23%. Among the drug coated stents theGroup 1 mean late loss value was 0.53 millimeters and the mean diameterstenosis was 11%. The Group 2 mean late loss value was 0.42 millimetersand the mean diameter stenosis was 11%. The Group 3 mean late loss valuewas 0.49 millimeters and the mean diameter stenosis was 12%. The Group 4mean late loss value was 0.28 millimeters and the mean diameter stenosiswas 10%. The significant reduction in both angiographic late loss anddiameter stenosis values for all four of the drug coated groups withrespect to expected values for the uncoated stents indicates a reductionin vessel restenosis due to the effect of the drug.

Trial B

Another set of in-vivo evaluations was designed to compare the effectsof two different drugs coated onto magnesium stents. The devices wereimplanted in porcine coronary arteries using a balloon to artery ratioof 1.15:1.00. These magnesium stents were first coated with parylene C,a permanent, durable, elastic polymer. The stents were then coated witha specific coating matrix comprised of a drug and a polymer. By firstapplying the parylene coating the magnesium features of the stent areassumed to be protected from corrosion for periods of time which exceedthe time required to deliver drug from the drug and polymer matrix.Group 1 of stents was coated with a matrix comprised of the drugrapamycin and polylactic acid such that the total dosage of the drug was223 μg. Group 2 and Group 3 of stents were coated with a matrix ofpolylactic acid and the drug rapamycin 40-O-cyclic hydrocarbon estersuch that the total dosage of that drug was 160 μg. All of the stentswere mounted onto balloon delivery catheters which were then packagedand sterilized with ethylene oxide gas. Stents in each of the threeseparate groups were deployed into one of three porcine coronaryarteries to a diameter of approximately 3.0 mm. Determination of theangiographic late loss and the percent diameter stenosis for eachimplanted vessel was made at 28 days post-implant. The results arelisted in Table X below.

TABLE X Angiographic Drug Weight Drug Molar Late Loss Diameter DrugAmount (μg) Amount(pM) (mm) Stenosis (%) Rapamycin - 223 240 0.42 13.1Group 1 (n = 5)* 40-O-cyclic 160 150 0.17 11.4 hydrocarbon ester - Group2 (n = 2)* 40-O-cyclic 160 150 0.24 11.9 hydrocarbon ester - Group 3 (n= 5)* *n = number of stents

Although the drug dosage of the rapamycin 40-O-cyclic hydrocarbon estergroups 2 and 3 was almost 30% lower by weight and 37.5% lower in molarconcentration than the drug dosage of the rapamycin group 1 there weresignificant reductions in both angiographic late loss and diameterstenosis values for both of the rapamycin 40-O-cyclic hydrocarbon estergroups compared to those values for the rapamycin group. This datademonstrates that with a lower dose the drug rapamycin 40-O-cyclichydrocarbon ester is more effective in treatment of vascular restenosisthan rapamycin.

IV. COMPOSITIONS AND APPLICATIONS IVA. Compositions

In another aspect, the invention includes a composition incorporatingthe rapamycin 40-O-cyclic hydrocarbon rapamycin ester of the invention,where the composition serves a drug reservoir for release, typically ina controlled fashion, of the compound into a treatment site.

Micro and Macro Particles

One exemplary composition includes a suspension of polymer particlesthat can be introduced into a suitable treatment site in vivo viainjection or delivery through a catheter. The polymer particles can bemicroporous, macroporous, or non-porous and can be formed of a polymerthat acts as a suitable drug reservoir. Exemplary polymer particles aredescribed, for example, in U.S. Pat. No. 5,135,740, incorporated byreference herein. Liposomal particles that incorporate the compound inencapsulated or membrane-entrapped form are also contemplated. Polymerssuitable for particle formation include, but aren't limited to poly(d,1-lactic acid), poly(l-lactic acid), poly(d-lactic acid), poly (glycolicacid) and copolymers and mixtures of polylactate and polyglycolic. Othersuitable polymers include methacrylate polymers, such as polybutylmethacrylate, ethylene vinyl alcohol (EVOH), .epsilon.-caprolactone,glycolide, ethylvinyl hydroxylated acetate (EVA), polyvinyl alcohol(PVA), polyethylene oxides (PEO), polyester amides, and co-polymersthereof and mixtures thereof. Typically, the composition is formulatedto contain between 20-80 weight percent polymer and 20-80 weight percentrapamycin compound. An exemplary formulation contains between 20 and 60weight percent of the rapamycin 40-O-cyclic hydrocarbon ester and 40 and80 weight percent of a polylactate or polyglycolate polymer or apolylactate/polyglycolate copolymer or mixed polymer. The particles havetypical sizes between about 0.1 micron to about 100 microns in diameter,preferably from about 0.5 microns to about 20 microns, and can beadministered as liquid, paste, or gel suspension.

Drug-Coated Stent

In another general embodiment, the invention includes an endovascularstent coated with the drug alone or the drug formulated in a polymercoated, such as the drug-polymer formulations described above. Suchstents are typically cylindrical, expandable metal-frame structureswhose struts or filaments may be coated, on their outer surfaces, with adrug-eluting coating. When placed in a body lumen, a stent is expandedto press against the wall of the vessel and hold the stent in place,while drug is eluted from the stent against the wall of the vessel.

An exemplary coating contains equal amounts by weight of compound 1 andd,l PLA material, which may be formulated and applied to the stent outersurface according to well known methods. An example stent product isdescribed in U.S. Pat. No. 7,214,759 where polymers containingpolyesters and optional therapeutic agents are applied to implantablesubstrates, including stents. Coatings applied to the surface of stentsmay incorporate excipient materials along with the rapamycin 40 O-cyclichydrocarbon esters which enhance the adhesion properties or elutionprofile of the drug, or impart other beneficial properties to thesystem. The coating may contain or be composed of micro or macroporousdrug reservoirs. The coating may be comprised of the drug compoundalone. Those familiar with the art are aware that these examples do notlimit the scope of the invention.

IVB. Treating Restenosis: Compound Delivery from a Catheter Balloon

In another vascular application, rapamycin 40 O-cyclic hydrocarbonesters can be applied as a coating to the surface of the inflatableballoon portion of a percutaneous vascular angioplasty balloon catheterin a manner that when the balloon is placed adjacent to a vasculartarget lesion and then inflated the drug will separate from the balloonsurface and transfer to the affected tissue. In this method of drugdelivery the balloon distributes the drug homogenously along thearterial wall, whereas in drug-eluting stents the highest concentrationis under the wire components of the stents. Whereas stents generallypermanently alter the vessel wall the drug coated balloon is removedafter deployment, leaving only the remaining drug and coating. In U.S.Pat. No. 7,572,245 Herweck, et al. describe a means to deliver atherapeutic agent a vessel lumen by positioning a coated balloon withina target vessel and through inflation of the balloon atraumaticallysmearing the coating agent against the vascular lesion, thustransferring a portion through lipophilic absorptive action. In U.S.Pat. No. 7,750,041 Speck, et al. reveal an angiography cathetercomprising a coating of paclitaxel and iopromide applied to the balloonportion of the catheter which is released to surrounding tissue uponballoon inflation.

Coatings applied to the surface of vascular balloons may incorporateexcipient materials along with the rapamycin 40 O-cyclic hydrocarbonesters which enhance the adhesion properties or elution profile of thedrug, or impart other beneficial properties to the system. The coatingmay contain or be composed of micro or macroporous drug reservoirs. Thecoating may be comprised of the drug compound alone.

IVC. Treating Ocular Disorders

Compounds in the subject invention may be used to treat oculardisosorders. Rapamycin itself has been evaluated clinically fortreatment of macular degeneration. In clinical trials subconjunctivalinjections of rapamycin in doses ranging from 220 micrograms to 880micrograms to verify increased visual acuity in a 180 day timeframe.Rapamycin has also been applied systemically in conjunction withcorticosteroids for treatment of non-infectious uveitis. The compoundsof the subject can be utilized in valved or non-valved stents/shuntsused for glaucoma drainage In the present invention, the oculardisorders are treated by administering to a mammalian subject in need oftreatment, a therapeutic amount of the rapamycin alkyl ester analog ofthe present invention.

Example I: Comparative Studies Between Sirolimus and Present Compoundsin Rabbit Vitreous and Aqueous Humour

In vitro studies were performed with New Zealand White rabbit vitreoushumour (which is an acceptable predicate for human vitreous humour) andintravitreal administration of the rapamycin 40-O-cyclic hydrocarbonester compounds of the present invention was varied by dosing andmeasured against sirolimus. The assay comparison between sirolimus andthe compounds of the present invention (denoted as CRC) is furtherdescribed at Table XI.

TABLE XI Assay comparison of sirolimus (SIR) drug stability in rabbitvitreous and aqueous humour at 37° C. versus CRC Drug Drug RemainingRemaining Drug Dose 72 Hours % 192 Hours % Trial (μg) (μg) Remaining(μg) Remaining Rabbit Vitreous Humour SIR 1 48.4  4.0 8% — — SIR 2 47.8— — 0.6  1% CRC 1 52.2 28.0 54%  — — CRC 2 54.0 — — 24.4  45% CRC 3261.8 — — 235.2  90% Rabbit Aqueous Humour SIR 1 45.2  0.0 0% — — CRC 151.4 34.4 67%  — —

As evidenced in Table XI, there is a much larger amount of CRC available(versus SIR) present over time, in both the vitreous humour and aqueoushumour. This is due to the fact that there is improved solubility of therapamycin 40-O-cyclic hydrocarbon ester compounds of the presentinvention (embodied in CRC), when compared to sirolimus. This allows fora lower injection volume for a dose size equivalent to sirolimus or, ifusing the same injection volume of drug, a higher dosing can bedelivered. This is particularly important when dealing withadministration directly to the vitreous, as intravitreal treatmentinjection volume is necessarily restricted to less than 50 μl and isgenerally within the 20-25 ul range or preferably less.

Additionally, CRC has much higher intravitreal chemical stability, whichmay allow for a longer interval between eye injections, as the druglevels are maintained for much longer time periods when compared tosirolimus. As shown at Tables XII and XIII, sirolimus is much morerapidly degraded over a shorter period of time when compared to CRC atboth the vitreous humour and aqueous humour samples. This is furtheridentified in reverse phase HPLC studies comparing the breakdown of SIRand CRC after 72 hours in rabbit vitreous humour (FIG. 3).

TABLE XII Comparison of detected breakdown materials after incubation inrabbit vitreous humour at 37° C. Rabbit Vitreous Humour (HPLC UV AreaPercent) Time Sirolimus CRC (hrs) Degradants Sirolimus Degradants CRC 0 3% 97%  4% 96% 1  3% 97%  5% 95% 4  7% 93% 14% 86% 24 43% 57%  6% 94%48 71% 29% 15% 85% 72 84% 16% 32% 68%

TABLE XIII Comparison of detected breakdown materials after incubationin rabbit aqueous humour at 37° C. Rabbit Aqueous Humour (HPLC UV AreaPercent) Time Sirolimus CRC (hrs) Degradants Sirolimus Degradants CRC 0 2% 98%  1% 99% 1  4% 96%  2% 98% 4 18% 82% 14% 86% 24 86% 14% 11% 89%48 96%  4% 16% 84% 72 100%   0% 22% 78%

U.S. Pat. No. 8,367,097 discloses liquid formulations containingrapamycin, polyethylene glycol and ethanol for the treatment of uveitisby intravitreal injection in humans. It has been shown that polyethyleneglycol can cause severe ocular toxicity when injected intravitreally.(Patel et al., J. OculPharmacol Ther 32(2):97-101 (2016).

Another advantage of using the rapamycin 40-O-cyclic hydrocarbon estercompounds of the present invention over sirolimus is the ability toformulate CRC compounds in the absence of polyethylene glycol, resultingin a much cleaner chemistry, including only the active compound andethanol (as opposed to sirolimus, which requires a solubilizing agent inthe formulation such as PEG to allow for proper drug concentrationcontained in suitable injection volumes.). Similarly, as the CRCcompounds are more lipophilic than sirolimus, there is a greater abilityto penetrate into the target tissues, resulting in an increased potency.

One aspect of the invention is therefore directed to a pharmaceuticalformulation comprising or consisting of CRC compounds as defined hereinand ethanol. In a preferred embodiment of the formulation theconcentration of CRC compounds as defined herein in ethanol is in therange of 1-wt-% to 60 wt-%, more preferred 10 to 57.5 wt-%, even morepreferred in a range of 20- to 55 wt-% and most preferred in a range of33.7 to 50.5 wt-%.

Also, the invention is directed to the use of such a pharmaceuticalformulation for the treatment of ocular disorders such as uveitis orproliferative ophthalmic diseases as described herein. Also, theinvention is directed to a dosage form comprising or consisting of avolume of the formulation as suggested herein.

IVD. Treating Cancers

Compounds in the subject invention are also useful in the treatment ofcancer. Rapamycin itself is currently in clinical pancreatic cancertrials as well as skin cancer trials and breast cancer trials. Thetherapeutic effect of rapamycin and related compounds in cancer andmalignant tumor treatment is likely caused by disruption of thebiochemical pathways involved in the development of new blood vessels.Rapamycin when combined with cancer drug Carboplatin have also beenfound to potentially improve the efficiency of ovarian cancer treatment.Rapamycin has also been shown to inhibit the progression of dermalKaposi's sarcoma. Other mTOR inhibitors, such as temsirolimus andeverolimus are currently being tested for use in cancers such asglioblastoma multiforme and mantle cell lymphoma. In the presentinvention, a cancer is treated by administering to a mammalian subjectin need of treatment, a therapeutic amount of the rapamycin 40-O-cyclichydrocarbon ester analog of the present invention.

Example II: Cell Culture Inhibition Studies for CRC Compounds

In vitro mammalian cell culture inhibition studies are frequently usedto predict biological in vivo responses. Various human cancer cell typeswere obtained from the American Type Culture Collection (ATCC) andcultured according to the vendor's recommendations. CRC was dissolved indimethyl solfoxide (DMSO) and added to the culture medium atconcentrations indicated. Controls consisted of DMSO only addition. Cellproliferation was measured using methylene blue staining by conventionalmethods.

The cell lines and the inhibition activity (IC₅₀) in the presence of CRCare described in the figures as follows: human breast cancer cell lineMCF7; CRC IC₅₀=81 nM (FIG. 4), human breast cancer cell line BT549; CRCIC₅₀=8 nM (FIG. 5), human melanoma cell line A375; CRC IC₅₀=11 nM (FIG.6), human colorectal adenocarcinoma cell line HT29; CRC IC₅₀=1 nM (FIG.7), human colorectal adenocarcinoma cell line WiDr; CRC IC₅₀=107 nM(FIG. 10 8), pancreas ductal adenocarcinoma cell line PANC-1; CRCIC₅₀=15 nM (FIG. 9), human pancreas ductal adenocarcinoma cell lineMiaPaCa2; CRC IC₅₀=252 nM (FIG. 10).

As evidenced in the data mentioned above, the inhibition of severalhuman cancer cell types by the rapamycin 40-O-cyclic hydrocarbon estercompounds of the present invention at low nanomolar concentrationsindicates utility of this material for oncology treatments. The presentdisclosure provides a method of treating cancer comprising administeringan effective amount of the rapamycin 40-O-cyclic hydrocarbon estercompounds of the present invention at a nanomolar dosage range and aform of administration that delivers the compounds in a pharmaceuticallyacceptable form. Dosage forms well known in the art such as but notlimited to pills, injections, creams, suppositories, depots, solutions,balloons and stents can be utilized to dose a mammal in need oftreatment.

IVE. Prevention of Transplant Rejection

Rapamycin and its analogs can be used alone, or in conjunction withcalcineurin inhibitors, such as tacrolimus to provide immunosuppressionregimens. Transplant patients can be given oral medications whichinclude rapamycin under the trade name Rapamune® or everolimus, arapamycin analog under the trade name Certican® to reduce the incidenceof tissue rejection. These therapies have the advantage of having lowertoxicity toward kidneys. In the present invention, the subject istreated by administering a therapeutic amount of the rapamycin alkylester analog of the present invention.

Although the invention has been described with respect to particularembodiments and applications, it will be appreciated that variousadditional modification the applications may be made consistent with thescope of the claims.

We claim:
 1. A method of treating a disease selected from the groupconsisting of restenosis, wound healing, vascular injury, vascularinflammation, transplantation rejection, proliferative ophthalmicdiseases (including wet age-related macular degeneration (AMD) anddiabetic macular edema (DME)), uveitis and cancer in a mammal byadministering an effective amount of a 40-O-cyclic hydrocarbon estercompound having the structure:

where R is C(O)—(CH₂)_(n)—X, n is 0, 1 or 2, X is a cyclic hydrocarbonhaving 3-8 carbons, optionally containing one or more unsaturated bonds,one or more linear-chain or cyclic carbon atoms may contain an OH orhalide substitution, and the rapamycin parent structure may containsubstitutions at non-40 positions.
 2. The method of claim 1, wherein the40-O-cyclic hydrocarbon ester compound is administered in the form of aformulation comprising or consisting of ethanol and the 40-O-cyclichydrocarbon ester compound.
 3. The method of claim 1, wherein the40-O-cyclic hydrocarbon ester compound is administered in the form ofpills, injections, creams, suppositories, depots, solutions, balloonsand stents to dose a mammal in need of treatment.
 4. A pharmaceuticalformulation comprising ethanol and 1-60 weight percent the rapamycin40-O-cyclic hydrocarbon ester of claim
 1. 5. The formulation of claim 4consisting of ethanol and the rapamycin 40-O-cyclic hydrocarbon ester.6. A dosage form comprising or consisting of a volume of liquid of claim4 or
 5. 7. A method of treating uveitis comprising administering aneffective amount of a 40-O-cyclic hydrocarbon ester compound having thestructure:

where R is C(O)—(CH₂)_(n)—X, n is 0, 1 or 2, X is a cyclic hydrocarbonhaving 3-8 carbons, optionally containing one or more unsaturated bonds,one or more linear-chain or cyclic carbon atoms may contain an OH orhalide substitution, and the rapamycin parent structure may containsubstitutions at non-40 positions, wherein the 40-O-cyclic hydrocarbonester compound is combined with ethanol at a concentration range betweenat least 1 wt % and at least 60 wt %.
 8. A method of treating cancercomprising administering an effective amount of a 40-O-cyclichydrocarbon ester compound having the structure:

where R is C(O)—(CH₂)_(n)—X, n is 0, 1 or 2, X is a cyclic hydrocarbonhaving 3-8 carbons, optionally containing one or more unsaturated bonds,one or more linear-chain or cyclic carbon atoms may contain an OH orhalide substitution, and the rapamycin parent structure may containsubstitutions at non-40 positions, wherein the 40-O-cyclic hydrocarbonester compound is administered to an individual in need thereof at adose range of the compound between about 0.1 nM to about 400 nM.